Title: Peripheral Immune Development in Premature Infants with and without NEC Abstract: Necrotizing enterocolitis (NEC) is a devastating complication of prematurity that frequently results in death (20- 50%) or severe systemic complications. Although NEC is a multifactorial disease, its precise etiology continues to be poorly understood. As such, there are still no effective prevention methods or treatments available. Given increased intestinal and systemic inflammation seen in NEC, it is likely that the dysregulation of the immune system contributes to its pathogenesis. In order to better understand NEC pathogenesis, it is essential to have a solid understanding of normal immune development that occurs over the first two months of age in premature infants. Our preliminary data obtained from fetuses, patients with NEC and control subjects, show that there is a drastic reduction of total and tissue resident memory T cells in the intestinal tissue and an increase in T cells in the periphery. Moreover, NEC is associated with a significant increase in pro-inflammatory macrophages in intestinal tissue, and a concomitant decrease in circulating monocytes. Recent work has begun to address how neonatal peripheral immunity develops. However, data on the peripheral immune development in premature infants over the first two months of life is sparse. Therefore, the goal of this exploratory proposal is to define normal immune development of premature infants over the first two months of life and characterize its dysregulation in patients with NEC. The overarching hypothesis of this proposal is that development of the peripheral immune system in preterm infants is different in the innate and adaptive immune compartments from that of term infants, and it is this difference that makes them susceptible to NEC. As the blood volume in premature infants is limited, we have developed a methodology to perform deep immunophenotyping on as little as 100 liters of blood using mass cytometry time of flight (CyTOF). This unique methodology will allow us to address the hypothesis, with the following aims: 1) Define the peripheral immune development of premature infants (<32 weeks) over the first two months of life. We will perform CyTOF on peripheral blood from 50 premature infants, with samples collected at 0, 1, 2, 4, and 8 weeks. Deep characterization of the immune cell landscape and maps of the developmental trajectories of the major cell lineages in premature infants will be generated. 2) Identify peripheral immune cell abnormalities associated with the development of NEC. We will identify dysregulation of immune population trajectories specific to premature infants who develop NEC. This information will provide us with a model for the immune cell mechanism underlying susceptibility to NEC. This project would represent the first detailed deep examination of the development of the immune system in premature infants with and without NEC over the first two months, and will provide sufficient data for the development of a large-scale, in-depth study of the mechanism underlying the development of NEC.